Enzymes and Restriction

Question 1

What are 3 recombinant proteins that are made using genetic engineering?

Answer 1

→ Insulin
→ Interferon- involved in antiviral defence
→ G-CSF- promotes formation of bone marrow.

Question 2

What is G-CSF?

Answer 2

Produced by infected cells and induces antiviral defense

Question 3

What do nucleases do?

Answer 3

Degrade nucleic acids by hydrolysing phosphodiester bonds

Question 4

What does ribonuclease degrade?

Answer 4

RNA

Question 5

What does deoxyribonuclease degrade?

Answer 5

Degrades DNA

Question 6

What do exonucleases degrade?

Answer 6

Degrades from the end of the molecule

Question 7

What do endonucleases degrade?

Answer 7

Degrades from the middle of the molecule

Question 8

What is the purpose of restriction?

Answer 8

To limit the transfer of nucleic acids from infecting phages into bacteria

Question 9

What 2 things do restriction endonucleases do?

Answer 9

→ Recognise a specific sequence
→ Cut a specific sequence

Question 10

What sequence does ECOR1 recognise?

Answer 10

GAATTC

Question 11

What are the characteristics of recognition sites?

Answer 11

→ 4-8 base pairs in length
→ they are palindromic

Question 12

How often does a 4 base sequence occur?

Answer 12

Every 265 bases

Question 13

When does a 6 base recognition sequence occur?

Answer 13

Every 4096 base pairs

Question 14

What 2 nucleases produce an overhang?

Answer 14

→ EcoR1
→ Kpn1

Question 15

What nuclease does not produce an overhang?

Answer 15

Alu 1

Question 16

How are DNA fragments separated in electrophoresis?

Answer 16

By charge

Question 17

Where does the DNA move during electrophoresis and why?

Answer 17

→ DNA is negatively charged due to the phosphate groups
→ when a current is applied the DNA moves to the +ve pole

Question 18

What is the relationship between migration and fragment size?

Answer 18

Smaller fragments run quicker

Question 19

How do you find out the size of the fragment?

Answer 19

You compare the digested fragments to the standard ones to compare the length

Question 20

What does having two bands in a linear fragment mean?

Answer 20

It means you have two restriction sites

Question 21

What kind of a mutation occurs in sickle cell?

Answer 21

→ A single point mutation
→ Instead of GAG you get GTG

Question 22

What effect does the mutation have in sickle cell?

Answer 22

Induces changes in the beta globin gene which makes it defective

Question 23

What is the restriction site for the enzyme DDE1?

Answer 23

CTGAG

Question 24

How do you test for sickle cell using DDE1?

Answer 24

→ Purify DNA
→ Fragment of 450 bp
→ Digest the PCR with DDE1
→ If there is normal beta globin - 2 restriction sites and 3 fragments
→ If there is sickle cell beta globin - 1 restriction site and 2 fragments

Question 25

How can DNA molecules from different sources be joined together?

Answer 25

→ ECOR1 overhangs are compatible
→ Two fragments are mixed with DNA ligase and a recombinant molecule is produced
→ DNA ligase makes covalent phosphodiester bonds between DNA fragments

Question 26

What does DNA polymerase do?

Answer 26

Copies DNA based on a template

Question 27

Why do you use DNA polymerase?

Answer 27

→ PCR amplification
→ Generations of probes
→ Blunt ending of DNA overhangs

Question 28

How does phosphatase work?

Answer 28

Hydrolyzes a phosphate group off its substrate

Question 29

What two types of phosphatases are used?

Answer 29

→ Calf intestinal phosphatase
→ Shrimp alkaline phosphatase

Question 30

Why do you use a phosphatase?

Answer 30

→ To prevent cut plasmids from resealing
→ If the phosphate group in the plasmid isn’t removed it can reseal
→ If it is removed then it cannot be resealed alone because DNA ligase needs a phosphatase group to work on one single size

Question 31

What does polynucleotide kinase do?

Answer 31

→ Converts phosphate from ATP to substrate
→ Adds phosphate to the 5’ hydroxyl group of DNA or RNA

Question 32

Why would you use a polynucleotide kinase?

Answer 32

→ To phosphorylate chemically synthesized DNA so that it can be ligated to another fragment

→ To sensitively label DNA so that is can be traced using radioactive or fluorescently labelled ATP

Question 33

What is a probe?

Answer 33

→ fragment of ssDNA
→ 20-1000 bases in length
→ Complementary to the gene of interest

Question 34

What are reverse transcriptases isolated from and why?

Answer 34

→ Isolated from RNA containing retroviruses - HIV
→Not found in eukaryotes

Question 35

What do reverse transcriptases do?

Answer 35

Synthesize DNA molecule complementary to a mRNA template using dNTPs

Question 36

What is needed to form a DNA copy from RNA?

Answer 36

A primer

Question 37

What is a random primer?

Answer 37

→ cDNA upto 700bp
→ covers all the length of the RNA molecules

Question 38

What is an oligo dT primer?

Answer 38

→ Useful for cloning cDNAs and cDNA libraries but some might not be full length

→ a TTT sequence is used because genes that code for proteins are polyadenylated

Question 39

Why are specific primers designed for reverse transcriptase?

Answer 39

Reverse transcriptase has a size limit

Question 40

What does palindromic mean?

Answer 40

Reading in a certain direction on one strand is identical to the sequence in the same direction on the complementary strand

Question 41

Why do bacteria produce restriction enzymes?

Answer 41

→ Work as defence system for bacteria from bacteriophage

→The restriction enzymes cleave the phage DNA

Question 42

Why do restriction enzymes not cleave their own DNA?

Answer 42

Because of methylation of bacteria DNA so not recognised by restriction enzymes

Question 43

How do phosphatases prevent resealing?

Answer 43

Remove 5’-phosphate groups from DNA, RNA and both ribo and deoxy-ribonucleoside triphosphates

Question 44

Blunt ends or sticky ends, which has a lower efficacy ligation?

Answer 44

Blunt ends

Question 45

In SCA, what restriction site is lost?

Answer 45

DdeI site (5’CTNAG3’)